The present invention is directed to streptavidin molecules having secondary functional domains such as a cell adhesion domain.
Streptavidin, a protein produced by Streptomyces avidinii, forms a very strong and specific non-covalent complex with the water soluble vitamin biotin. Streptavidin is a tetrameric protein that binds biotin with an affinity that is among the highest displayed for non-covalent interactions between a ligand and protein, with an association constant (Ka) estimated to be in the range of 1013Mxe2x88x921 to 1015Mxe2x88x921. This binding affinity is strong enough to be essentially irreversible under normal physiological solution conditions, and provides the basis for streptavidin and biotin""s usefulness in a wide variety of clinical and industrial applications. See, Green, Adv. Prot. Chem. 29:85-143 (1975).
Both streptavidin and the homologous protein avidin, which shares its high affinity for biotin, have been studied as paradigms of strong ligand-protein interactions. The X-ray crystal structures of streptavidin and avidin, both in their apo and holo forms, have been described. The sequences of both have also been reported, as have the construction of several streptavidin fusion proteins (Sano and Cantor, Biochem. Biophys. Res. Commun. 176:571-577 (1991); U.S. Pat. No. 4,839,293).
In addition to the extremely high binding affinity, the usefulness of streptavidin also arises from the unique architectural properties of the protein. Streptavidin is a tetramer of four identical subunits, with each subunit contributing a binding site for biotin. Because the tetramer has approximate two-fold symmetry, the binding sites are positioned in pairs on opposite sides of the molecule, making the protein an efficient molecular adaptor. This structural feature, along with the high affinity of streptavidin for biotin, has made the protein an important component in many technologies.
Streptavidin is a key components in four technological areas of great significance: 1) bioseparations/cell sorting; 2) imaging; 3) drug delivery; and 4) diagnostics (Wilchek and Bayer, in Meths. Enzymol. 184:5-45 (1990)). In the separations area, this protein has been used extensively in important cell sorting applications, where, for example, it is used to remove contaminating cells from hematopoietic stem cells prior to marrow transplantation. Berenson et al., Prog. Clin. Bol. Res. 377:449-459 (1992). Streptavidin has found similar wide use in cancer diagnostics, where it is used in both research and clinical settings to test for the presence of various tumor specific biomarkers.
The imaging and drug delivery applications of streptavidin and biotin arise from the capability for simultaneous targeting and delivery of imaging agents or therapeutics to tumor cells. There is particularly significant emerging interest in the use of streptavidin for targeted delivery of imaging agents and therapeutics in vivo. Streptavidin has been used to deliver drugs, toxins and imaging agents to targeted cells both in vitro and in vivo. See, for example, Meyer et al., Exp. Hematol. 19:710-713 (1991). In these systems, streptavidin plays the crucial role of molecular adaptor between an antibody that serves as the targeting component, and a biotinylated therapeutic or imaging agent. With some strategies, cells are pre-targeted with the antibody-streptavidin conjugate, with subsequent delivery of the biotinylated agent. In other applications, a biotinylated antibody is first used to pre-target cells, with subsequent delivery of the streptavidin-biotinylated agent conjugate. A three-step delivery is also possible, using biotinylated antibody followed by streptavidin and then the biotinylated agent.
It would be advantageous to have a streptavidin molecule that contains a secondary functional domain so that biotin binding is retained but the streptavidin molecule also has another function. For example, it would be advantageous to have a streptavidin molecule that binds to a cell so that, for example, molecules can be selectively delivered to that cell. It would be advantageous to have streptavidin that selectively binds to a particular cell type or to a particular protein so that selective purification can be performed.
Streptavidin molecules are disclosed that contain a secondary functional domain. In preferred embodiments, the secondary domain is a cell adhesion peptide incorporated in the streptavidin amino acid sequence at a site not interfering with biotin binding. In a preferred embodiment, the cell adhesion peptide is arginine-glycine-aspartate (Arg-Gly-Asp) (RGD). The peptide is preferably placed on an exposed loop of the streptavidin molecule, such as within the loop defined by residues 63 to 69. The mutant streptavidin molecule can have other characteristics such as reduced biotin binding due to a modification of an amino acid at the biotin binding site. Preferred uses for the disclosed streptavidin molecules are as adaptors to bring, via a streptavidin/biotin interaction, the secondary functional domain into proximity with a cell or molecule to be affected and as a coating for substrates such as vascular devices or prostheses.